【作者】 刘绪立；

【导师】 雷伟；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 一、研究背景骨质疏松症最常见的并发症是椎体压缩骨折,手术治疗包括脊柱内固定和椎体成形术或球囊扩张椎体成形术。椎体成形术是近年来发展起来一种新的脊柱微创手术,该技术已广泛应用于治疗椎体的多种良恶性病变及骨质疏松性骨折,取得了很好的临床疗效,可以快速缓解疼痛,纠正脊柱畸形,由于椎体成形术创伤小、效果好且疗效快,已成为脊柱微创介入治疗的又一重要方法。临床上现在最常用的椎体成形术的材料为聚甲基丙烯酸甲酯(PMMA)。但PMMA充填材料具有许多缺点:病变椎体术后与相邻椎体的力学强度不同,因应力集中易导致相邻椎体的骨折;组织相容性差,无成骨作用,无生物降解性,最终不被自体骨取代;单体有细胞毒性,放热反应,其体外聚合温度达40～122℃,术中可能会引起邻近组织和神经元的热损伤。而且临床越来越要求成形材料能够携带有特殊治疗作用的药物,而PMMA并不能满足这些临床治疗的需要。如何探索一种具有良好生物相容性的成形材料,其在作为固定材料的同时又能成为携带具有特殊治疗作用的药物的载体,是本研究的目的。二、研究目的1.探索采用去势法+甲强龙诱导法快速建立兔子骨质疏松模型的方法;评估去势+甲强龙诱导后松质骨及皮质骨的微观结构和宏观力学性能的改变;从而建立一种适用于骨科领域研究的兔骨质疏松模型的快速建模方法。2.在已经建立的骨质疏松模型体内,采用“二维组织形态—三维空间结构—生物力学特性”的综合手段,评估硫酸钙以及硫酸钙复合bBMP对提高骨质疏松椎体骨密度、骨小梁微观结构及力学强度的作用。三研究内容和方法第一部分建立并分析评价兔骨质疏松动物模型采用随机对照研究方案,将20只新西兰大白兔随机分为去势(OVX)A组(n=4)、B组(n=8)和假手术组(Sham,n=8)。A组为单纯OVX组,B组为OVX+甲强龙肌注(4周)组。所有动物术前和术后2月均采用双能X线吸收骨密度仪测定腰椎骨密度。于术后2个月:①采用组织病理学切片方法对骨小梁结构进行组织病理学观察;②采用DEXA测定所有动物股骨髁部及皮质骨的骨密度;③采用显微CT分析松质骨骨小梁三维结构改变;④采用生物力学实验,比较实验组和对照组的松质骨和皮质骨的力学强度。第二部分硫酸钙(calcium sulfate CSC)复合bBMP材料在椎体强化中的实验研究采用随机对照研究方案,将60只新西兰大白兔随机分为C1组、C2组、C3组、C4组(n=12)和假手术组(Sham,n=12)。C组按照实验一方法建立骨质疏松模型,实验分为假手术对照组(Sham),骨质疏松对照组(C1),注射PMMA组(C2),注射CSC组(C3),注射CSC/bBMP组(C4),通过模拟椎体成形术,分别在每只兔子L2、L4、L6椎体注射材料,每组均于术后24小时,6周、12周处死4只。①采用组织病理学对骨小梁结构进行组织病理学观察;②采用MicroCT对骨小梁微观三维结构进行分析;③采用生物力学实验评估椎体骨质的结构力学强度。通过“微观结构-宏观密度-功能强度”的方法,全面衡量硫酸钙和硫酸钙复合bBMP材料对脊柱骨质疏松的治疗作用。四研究结果第一部分:手术前,Sham组、OVX-A组和OVX-B组的腰椎BMD分别为(266.7±38.58)mg/cm2、(270.1±25.38)mg/cm2和(272.8±27.08)mg/cm2手术2个月后,Sham组、OVX-A组和OVX-B组的BMD分别为(281.8±39.22)mg/cm2、(248.9±26.14)mg/cm2和(199.9±30.76)mg/cm2。OVX-B组的BMD较术前平均下降为26.72%。组织学切片观察发现,去势2个月后,OVX-B组椎体及股骨松质骨骨小梁明显稀疏、变细,局部存在骨小梁骨折及骨缺损;而对Sham和OVX-A照组骨小梁结构完整,呈圆形或椭圆形拱形结构。MicroCT分析表明,OVX-B组的骨小梁三维构筑较Sham组及OVX-A组均有显著改变(骨小梁明显变细、连接率明显下降),而OVX-A组骨小梁结构与Sham组无显著性差别。生物力学研究显示,OVX-B组椎体抗压缩强度(6.8±2.02MPa)较Sham组(14.5±3.74MPa)和OVX-A组(12.8±3.12MPa)均有显著性降低,而OVX-A组与Sham组无显著性差异。第二部分:组织形态学观察发现,CSC组和CSC/bBMP组骨小梁连接性在术后6周和12周明显优于C1对照组,形态较为规则,小梁间连接程度优于对照组。骨小梁变粗,骨折、微骨缺损处出现连接和修复;12周时骨小梁结构已经接近Sham对照组。MicroCT三维重建显示,6周时, CSC组和CSC/bBMP组骨小梁组的骨小梁三维结构参数明显优于C1对照组(P<0.05),骨密度值也明显优于C1对照组(P<0.05);12周时,已与Sham对照组无显著性差别。生物力学实验结果表明,在6周时,CSC组椎体的最大抗压缩强度有增加,但与C1对照组相比无显著性差异,而CSC/bBMP组椎体抗压缩强度在6周时显著高于C1对照组;在12周时,CSC组和CSC/bBMP组椎体最大抗压缩强度均较C1对照组有显著性差异(P<0.05),与Sham组及PMMA组相比,未见显著性差异(P>0.05)。五结论1.通过去势+甲强龙肌注的方法,可以在2个月内快速建立兔的骨质疏松模型。2.兔骨质疏松模型建成后,二维及三维图像显示椎体松质骨内骨小梁稀疏、变细、断裂,微观三维构筑受到破坏,并且局部形成骨小梁微骨折及微骨缺损,最终导致其力学性能下降。是一种理想可用的骨质疏松动物模型。3.CSC以及CSC为载体复合bBMP的注射型成骨材料具有强大的骨诱导能力,可以迅速有效地改善骨质疏松椎体骨小梁的三维构筑,提高其椎体力学强度,是具有良好应用前景的椎体充填材料。更多还原

【Abstract】 BackgroundThe compression fracture in spine is the most complication of osteoporosis. The medical therapies and braces are the primary measures. The surgical measures include internal fixation, vertebroplasty and kyphoplasty. Vertebroplasty is a minimally invasive surgery in spine developed in recent years. It has got good clinical effects in the therapy of benign or malignant lesions and osteoporosis compression fractures in spine. It has become prevalent in minimally invasive surgery of spine for rapidly relieving the back-pain and correcting the deformity. Now, the most commonly used cement is polymethylmethacrylate (PMMA) in vertebroplasty. Unfortunately, PMMA has several potential disadvantages. PMMA is not bioabsorbable and remains permanently in the body. Its unreacted monomer is toxic, and its high polymerization temperature has resulted in temperature readings as much as 1220C. The high compressive strength and stiffness of PMMA causes a biomechanical mismatch between treated and untreated vertebral levels which may increase the risk of adjacent-level fractures. It has been required that the injecting materials can carry medicine for special treatments in clinic. So PMMA can not satisfy these demands. How to explore a biological injecting material,which can not only support the vertebrae but also carry medicine for special treatments, is the object of this experiment.Objective1. To study the approach of rapid establishment of an osteoporosis rabbit model through ovariectomy(OVX) and injecting solu-medrol and to evaluate the micro-architectural and biomechanical changes of cancellous and cortical bone after OVX. To establish a rapid osteoporosis animal model which can meet the requirements of orthopaedic research.2. To evaluate the effects of calcium sulfate cement (CSC) and CSC combined with bovine bone morphogenetic protein (bBMP) on osteoporotic rabbits vertebral micro-architecture, bone mineral density and biomechanical properties in vivo.Material and methodsPart 1 Establishment and evaluation of an osteoporosis rabbit model.20 adult rabbits were randomly divided into six groups: sham-operated group (n=8), OVX-A group (n=4) and OVX-B (n=8) groups. OVX-B group was started to inject solu-medrol for 4 consecutive weeks after 2 weeks late of OVX. Sham group and OVX-A group were control groups. Before and 2 months after the operation, all the rabbits of study underwent dual energy x-ray absorptiometry (DEXA) of the lumbar vertebrae twice. 2 months after the operation, the rabbits were killed. The femoral condyle and shaft underwent DEXA. The trabecular bone(spine and femoral condyle) and cortical bone (femoral shaft) were harvested and reconstructed by micro-CT. Compressive mechanical properties of cancellous bone were determined from biopsies of vertebral bodies. Three-point-bending tests were used to evaluate the mechanical properties of cortical bone (femur shaft).Part 2 the study on the vertebrae augmentaion in osteoporotic rabbit vertebrae with calcium sulfate (CSC) /bBMP composite.60 adult rabbits were randomly divided into five groups: sham-operated group (n=12), C1 group (n=12), C2 group (n=12), C3 group (n=12) and C4 group (n=12). C groups were estabilished osteoporotic rabbit models according to the methods of part 1 experiments. Sham group and C1 group were control groups. 0.1-0.2ml PMMA (Group C2), CSC (Group C3) and CSC/bBMP (Group C4) were injected into L2, L4 and L6 vertebra of every rabbits. 4 rabbits were killed respectively from every group in 24 hours, 6 weeks and 12 weeks after PVP. The trabecular architecture changes were observed histomorphologically. Micro-architecture of vertebrae was measured by micro-CT after the rabbits were killed. Axial compression tests were carried out on vertebral specimens to evaluate their strength.ResultPart 1: The pre-OVX BMD for lumbar spine in Sham, OVX-A and OVX-B were (266.7±38.58)mg/cm2、(270.1±25.38)mg/cm2 and(272.8±27.08)mg/cm2 respectively. 2 months later, the post-OVX BMD in Sham, OVX-A and OVX-B were ( 281.8±39.22 ) mg/cm2、( 248.9±26.14 ) mg/cm2 and(199.9±30.76)mg/cm2 respectively. BMD in OVX-B decreased by 26.72% compared with the pre-OVX BMD. Histomorphologically, trabecular bone decreased significantly in OVX-B group compared with that in sham group and OVX-B group. Micro-fracture and micro-defect could be seen in OVX-B.The parameters of trabecular bone had significantly difference compared with control groups (p<0.05). In biomechanical testing, the maximum compression strength of vertebrae in OVX-B was lower than that in the other two groups. In 3-point bending test, there was no difference between three groups.Part 2: Histomorphologically, the connectivity in group C3 and C4 was higher than that in C1 group respectively at 6th week and 12th week. The shape of trabecular bone was also better.The architecture of trabecular bone was same as that in sham group. Micro-fractures and micro-defects were also less than that in the C1 groups. After injection of CSC or CSC/bBMP, the Micro-CT reconstruction analysis showed that the BMD in group C3 and C4 were significantly higher than that in group C1 respectively at 6th week and 12th week(P<0.05). The trabecular thickness, connectivity and bone fraction in group C3 and C4 were significantly higher than that in C1 group and were as good as the sham group in 12th week. The results of biomechanical test showed that the maximum compression strength in C4 was higher significantly than that in C1 group in 6th week (P<0.05). It was also higher significantly in both C3 and C4 than that in C1 and had no significantly difference compared with that in sham group and C2 group in 12th week.Conclusion1. Osteoporosis rabbit model can be rapidly establisher by ovariectomy and injecting solu-medrol in 2 months.2. After an osteoporosis rabbit model is induced, the trabecular bone in vertebrae becomes thinning and sparsely. Micro-fractures and micro-defects occur and result in the decrease of biomechanical properties. It is an ideal osteoporosis model for experiments. 3. CSC and CSC/bBMP injecting in osteoporosis vertebrae can repair the micro-fractures and micro-defects in trabecular bone and can enhance the bone quality significantly. CSC and CSC/bBMP can be used as local treatment of vertebral osteoporosis.更多还原